Method for the separation of cell fractions

ABSTRACT

The present invention relates to a method for the separation of cells, in particular for the preparation of samples in tumor diagnostics. In particular, the present invention relates to a method for sample preparation for the detection of tumor cells of solid tumors in the course of the diagnosis for prognosis and stratification of therapy, comprising the destruction of cells that make this diagnosis more difficult or entirely impossible. Furthermore, the present invention relates to a kit for the preparation of samples and for the detection of the presence of the altered cells. Finally, the present invention relates to the use of the method and the kit in the diagnostics of altered cells such as tumor cells, and the performance of a PCR reaction to detect the tumor cells in body fluids and tissues.

The present invention relates to a method for the separation of cells,in particular for the preparation of samples in tumor diagnostics. Moreexactly, the present invention relates to a method for samplepreparation for the detection of tumor cells of solid tumors in thecourse of diagnostics for prognosis and stratification of therapy,embracing the destruction of cells that make this diagnosis moredifficult or entirely impossible. Furthermore, the present inventionrelates to a kit for the preparation of samples and for the detection ofthe presence of the altered cells. Finally, the present inventionrelates to the use of the method and the kit in the diagnostics ofaltered cells such as tumor cells, and the performance of a PCR reactionto detect the tumor cells in body fluids and tissues.

PRIOR ART

The separation of different cell populations is a central aspect in theanalysis of different cell populations. Very different methods are knownin the prior art for the separation of cells and these are mostly basedon different physical characteristics, or on differences in theexpression of certain molecules.

The detection of the presence of circulating and/or micrometastaticaltered cells such as tumor cells, in a mixture of cells of differentcell populations enables an early assessment of the prognosis of thepatient and a stratification for possible, so-called, adjuvant therapysteps.

To enable meaningful early diagnosis, it must be possible tospecifically detect the presence of an extremely low number of alteredcells in the sample to be analyzed.

Tumor cells and other altered cells virtually always have geneticalterations compared to their normal precursors. The alterations relateeither to the appearance of tumor-associated and characteristic geneexpression profiles and/or are characterized by specific gene mutations.The detection of the presence of the latter (for instance mutations inthe genes p53, BRCA1 & 2, APC and others) has not so far beentechnically possible at the DNA level with the necessary diagnosticsensitivity if the tumor cells are under-represented relative to normalcells in the sample.

In contrast, detection of the presence of altered cells, such as tumorcells, at the required sensitivity is possible with the amplification ofa so-called “tissue-specific mRNA expression” (molecular staging),provided that the following pre-requirements are met:

-   -   a.) The source organ of the tumor is known. Tissue-specific mRNA        markers can be defined, such as        -   i) Cytokeratins, that represent general epithelial markers,            and the presence of which remains detectable for malignant            tumors of epithelial origin (carcinomas).        -   ii.) So-called differentiation markers of different tissue            such as, for example, CEA (for large intestine carcinomas),            PSA (for prostate carcinoma), AFP (for hepatic carcinomas),            tyrosinase (for malignant melanoma) etc.    -   b.) The normal cells otherwise still present in the test sample        (normal blood leucocytes of a blood sample) do not express the        mRNA marker used as a target for molecular staging: conversely,        the mRNA markers that are expressed in normal cells are not        suitable for staging.

If the pre-requirements are satisfied, then the presence of roamingaltered cells, such as tumor cells, can be detected in a test sample viathe tissue-specific mRNA expression profile using suitable sensitivemethods, in contrast to specific-specific gene mutations, also within alarge excess of normal cells.

In technical terms, the detection is based on the in vitro production ofa cDNA copy of the target mRNA using reverse transcription, with asubsequent DNA polymerase chain reaction (RT-PCR). This detection ispossible with a high sensitivity and generally embraces a single tumorcell in 10⁶-10⁷ normal blood cells (i.e. 1 ml blood).

To date, however, severe restrictions have been placed on the use of theRT-PCR test. A large number of tissue-specific expressed genes have beendescribed that are, in principle, suitable for the detection of thepresence of altered cells derived from that tissue, for example tumorcells. The detection of their presence, however, is not alwayssuccessful for the following reasons, as a result of system-immanentspecial characteristics of such amplification systems, given below:

The analytical and diagnostic sensitivity of detection methods based onRT-PCR is very high. In other words, the method enables the reliabledetection of individual molecules in test systems and in clinical testmaterial. In addition, the analytical specificity is very high. In otherwords, any erroneous amplification of molecules, other than the desiredmRNA molecules, through so-called cross-hybridization of the primer, canbe ruled out with certainty under suitable conditions.

However, the diagnostic specificity is insufficient. This means thatpositive results are regularly found for the selected “tumor-associated”target mRNA in clinical samples from normal individuals or patients withnon-malignant disease. Non-malignant cells in the sample can thereforegive rise to positive RT-PCR results. Correspondingly, it is difficultto interpret the results in individual cases for diagnosis purposes.

The resultant low diagnostic specificity is the reason why the detectionof the presence of circulating tumor cells through RT-PCR has not beenwidely adopted for the diagnosis of minimal residual disease (Jung etal., EJCCCB, 1997, Jung et al., J Lab Med. 1999). The false-positiveresults, in fact, do not permit the use of this intrinsically veryadvantageous method.

The main reason for the insufficient diagnostic specificity is mRNAbackground expression (also referred to as illegitimate transcription orbackground transcription) due to the presence of normal cells in thetest sample (for example, white blood cells in blood and bone marrow).It has been shown that normal white blood cells can expresstumor-associated mRNA markers. This “illegitimate” expression has a lowvalue at the level of the individual cell, but results in a markedmeasurable signal because of the high number of thesenaturally-occurring cells in the sample, and thus leads to theaforementioned non-specific positive results. The pattern of backgroundexpression may be constitutive (Jung et al., Br. J. Cancer (1999)), orinduced, for instance in the course of an inflammation reaction (Jung etal., Br. J. Cancer, (1998)).

It is evident from the literature since then that the problem ofbackground expression substantially reduces the utility of the method.Correspondingly, various methods have been proposed to increasespecificity and exploit the physical or expression-specificcharacteristics of the cells to be separated, similarly to the usualseparation methods. These methods, however, frequently have substantialdiagnostic restrictions for their clinical utility.

The most commonly used method for separation of cell populations, forinstance from whole blood, is the so-called FICOLL density gradientcentrifugation. A large number of variants of the same principle existfor FICOLL gradients. This is based on an enrichment of mononuclearcells in which the presence of a tumor cell is then detected.

The FICOLL method cannot be standardized, i.e., it is of varyingeffectiveness in the recovery of separated cells. A main determinant ofthe sensitivity of the detection of the presence of tumor cells is thetechnical expertise of the investigator (Krüger et al., Clin. Chem.,2000). In addition, the recovery and enrichment of altered cells, suchas tumor cells, is uncertain because of their unpredictablesedimentation behavior. In summary, it is unclear whether tumor cellsare lost in the course of sedimentation, and if so how many, and whetherthe tumor cells in general behave sufficiently homogenously in terms oftheir specific density that they can always be separated together withmononuclear cells and are not sedimented in the discarded granulocytefraction. Recovery rates of between 10 and 70% are reported overall.

The second main principle of the enrichment of target cells isimmunobead enrichment. This method is based on the fact that alteredcells, such as tumor cells, can be selectively derived from a cellmixture through binding to paramagnetic particles. A pre-requirement forthis is the expression of a tumor-associated marker at a suitabledensity on the surface of the tumor cell. Paramagnetic particles, ontowhich an antibody directed against this marker has been bound, areadsorbed onto the target cells and are then enriched from the solutionthrough the use of a magnet. The successful binding of the antibodyrequires a sufficient density of the marker on the cell surface.

The immunobead enrichment method has two limiting pre-requirements:

1) The marker has to be a surface marker and is therefore dependent onthe tumor type. However, the heterogeneity of carcinomas is such thatthere is no uniformly high and suitable expression of the marker on allindividual tumors or their metastases. The number of molecules on thesurface required for successful enrichment, is essentially unknown.

2) To allow treatment of the cells in an intact state, the methodrequires the treatment of native test samples directly following thesampling procedure. A degradation of the sensitive mRNA has beenobserved within a period of hours after blood sampling (Gerhard et al.,J. Clin. Oncol. 1994 Apr; 12 (4): 725-9). The associated decrease inmRNA quality rapidly reduces the diagnostic sensitivity of a RT-PCRtest. It follows that both the sample preparation and the time factorfor the immunobead separation are of decisive importance for thedetection of the presence of individual tumor cells on the basis oftissue-specific mRNA. These pre-requirements are not met for routinediagnostic use.

The U.S. application Ser. No. 2002/0012931 describes a method for thedetection of the presence of circulating tumor cells, in which the cellsresponsible for false-positive signals in the detection of GC-C(guanylyl cyclase C) were identified as CD34-positive cells in theblood. According to the method described therein, these false-positivecells should be removed from the cell mixture through elimination, forinstance through immunobeads. The presence of the GC-C mRNA in theremaining cells is demonstrated thereafter. This method thereforeinvolves a negative selection of the altered cells, i.e., the cellsresponsible for the false-positive signal are separated out.

An imperative requirement for both of the methods outlined above is thatthe sample be treated further immediately after sampling to ensure thatthe integrity of the mRNA within the derived cells is maintained.

The aim of the present invention is to remove these restrictions, in thestandardized performance of the method as well. This applies, inparticular, to those cases where RT-PCR is to be used in the course ofroutine diagnostic procedures after the separation procedure. The methodaccording to the invention thereby has to satisfy the followingpre-requirements:

1) It must be possible to easily remove the cells (sub-populations) inthe test sample responsible for illegitimate expression.

2) The above-mentioned sub-populations to be separated and theirconstituents should be “removed” from the sample. The mRNA expression bythe background cells must be overcome through this removal.

3) It must be possible to perform the method with a minimum of effort atthe place of sampling in a reliable manner so that the method isstandardized. Standardized in this context means firstly that thesystems used for sampling must lend themselves to pre-fabrication. Onthe other hand, it is necessary for the sampling protocol to be devisedsuch that the method can be performed by different investigators withequal efficiency when proceeding in the same manner. By definition, itis not possible to use a technically non-standardized method (such asthe FICOLL density gradient or immunoseparation) for this purpose.

4) The method must not be linked to the necessity of maintaining complextechnical apparatus at the place of sampling, as it would then no longerbe suitable for broad usage on a routine basis.

5) It must be possible to perform the method rapidly enough to avoidendangering the integrity of the mRNA in the sample. If this is not thecase then the diagnostic sensitivity would be critically reduced.

BRIEF DESCRIPTION OF THE INVENTION

To satisfy the above requirements, the method according to the inventionfor separation of normal cellular sub-population(s)/fraction(s)—asdefined below—from the altered cells in a sample embraces the step ofincubating the mixture of normal cells and altered cells in a hypotonicsolution and destruction of fractions thereof.

The method is based on a novel concept of destroying one or moredifferent cellular sub-populations/fraction(s) responsible for theillegitimate expression of the mRNA marker under investigation in thesolution through hypotonic influence before the sample is processedfurther for analysis of the altered cells, in contrast to themethodology commonly adopted to date.

The method according to the invention can, furthermore, embrace the stepof collecting and recovering the non-eliminated cells. If the method isto be used for further analysis of the collected cells, then an analysisstep, for example a RT-PCR, is used after the recovery step.

It was found that incubation in a hypotonic solution results in adifferentiated destruction of cell fractions in the samples. Inparticular, altered cells, such as tumor cells, have a greaterresistance to the hypotonic conditions in the solution than cellpopulations normally present in the sample.

The method according to the invention can be used to detect the presenceof altered cells, such as tumor cells. One possibility is the use of themethod for the diagnosis of metastasizing cancer.

The invention provides for a kit for the detection of the presence ofaltered cells, such as tumor cells. This kit embraces a hypotonicsolution, or means of rendering the solution hypotonic, and primers fordetection of mRNA coding for a specific-specific marker. Furthermore,the kit preferably embraces a RNA-stabilizing solution, comprising ahighly-concentrated chaotropic salt.

This kit can be used to diagnose metastatic cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 a shows the detection of the presence of cytokeratin 20(CK20) and (PBGD) under different hypotonic conditions for normal blood.FIG. 1 b shows the detection of the presence of cytokeratin 20 (CK20)and (PBGD) under different hypotonic conditions for normal blood with acertain number of tumor cells.

FIG. 2: FIG. 2 shows the detection of the presence of cytokeratin 20(CK20) and (PBGD) under different hypotonic conditions for normal bloodwith 25 tumor cells (HT29).

FIG. 3: FIG. 3 shows the relative proportion of the granulocytesub-population within the blood cells as a function of the treatmentwith hypotonic solutions.

DETAILED DESCRIPTION OF THE INVENTION

The following terms, as used herein, are explained in more detail, toassist in understanding the application.

Osmolarity/osmolality: Osmolarity is the concentration ofosmotically-effective particles in 1 liter of test material (osm/L).Osmolality is the concentration of osmotically-effective particles in 1kg solvent (osm/kg). Milliosmoles are abbreviated to mosm.

Hypotonicity: Solutions of the same osmotic pressure are isotonic.Solutions of increased osmolality are termed hypertonic and solutionswith a lower osmolality as hypotonic.

Destruction of cells: The removal of cell integrity throughchemical/biological or physical methods. An example is the addition of ahypotonic liquid to cells to burst them.

Tumor-associated/specific mRNA: In the course of gene expression one ormore “copies” of a gene (DNA) is produced in the form of mRNA (messengerRNA).

This process is referred to as transcription. Thereafter, the geneticinformation of the mRNA is converted to an amino acid sequence (known astranslation). 3o This results in proteins that can assume multipleroles. Tumor-specific/associated RNA molecules are characteristic fortumors and are either not formed by normal cells, or frequently in smallquantities. The mRNA coding for a tumor-associated molecule is thus theprecursor of the tumor marker detectable by immunocytochemical methods.

Solid tumors: Solid tumors are distinguished from non-solid tumorsthrough the formation of a measurable, coherent tumor mass. Carcinomasare an example of a solid tumor and leukemia is an example of anon-solid tumor.

Stabilizing: In the context of the invention, stabilization is firstly aprevention of the degradation of RNA and, secondly, the protection oftumor cells against destruction by a hypotonic solution.

Cell constituents: Cell constituents comprise all structures, substancesand molecules that define the cell in its form and function. Theseinclude, for example, the cell nucleus, the cell membrane, thecytoplasm, DNA, RNA and proteins etc.

Cells: Cells in this context are all cells, in their entirety, that makeup a human being.

Cell types: The types of cells are, for example, blood cells or tumorcells, intestinal epithelial cells etc.

Cell populations: These are sub-groups within a cell type, such as whiteblood cells (leukocytes) and red blood cells (erythrocytes) within theblood cells.

Sub-populations: These are sub-groups within the cell populations, suchas, for example, granulocytes and lymphocytes within the white bloodcells (leukocytes).

Cell fractions: Cell fractions are sub-groups within thesub-populations, for example eosinophils and basophils within thegranulocytes.

Normal cells/non-tumor cells: These are all of the cells that are notmalignant or altered in any other way. In the present case, normal bloodcells are assumed. Altered cells, such as tumor cells or precursors oftumor cells, exist in contrast to these.

Background expression: Altered cells, such as tumor cells, express largequantities of certain marker molecules (for example tumor-associatedmRNAs such as CK20 mRNA, CEA mRNA or PSA mRNA) that are characteristicfor certain cell types such as epithelial cells. Some normal blood cellsexpress very low quantities of these mRNAs. The presence of a largenumber of normal blood cells in a sample will therefore generate asignal that corresponds to that of a low number of tumor cells producingthe marker molecule in large quantities.

This expression of marker molecules at a very low level by normal bloodcells is defined herein as background expression. Synonyms forbackground expression are illegitimate transcription and backgroundtranscription

CP value: The “Crossing Point” (CP value; threshold cycle) of a PCRreaction measured in a LightCycler® (Roche) is the PCR cycle at whichPCR amplification enters the exponential phase. What is meant is theexact time point of the PCR reaction (cycle number) at which thefluorescence of a certain reaction exceeds the background fluorescence.This time point is most reliably proportional to the concentration ofthe template present at the start of the amplification process. Thistime point is automatically established by the Roche LightCyclersoftware in graphical form.

The lower a CP value, the more copies of the nucleic acid section to beamplified are present in the sample. If no CP value is established, thena sample does not contain a target molecule that can be amplified.

Porphobilinogen Deaminase (PBGD): PBGD is a so-called “housekeepinggene” and is expressed constitutively at a low level by all somaticcells. It acts as a positive control for the presence of mRNA in thesample.

The invention provides a method for the separation of cell fractionscomprising normal cells and altered cells. The cell fractions comprisingnormal cells and altered cells are incubated in a hypotonic solution.One or more cell fractions are destroyed in this process.

The invention is based on the finding that cells differ in theirresistance to a hypotonic solution before cell integrity is destroyed.More exactly, it was found by the inventors that altered cells, such astumor cells, are more resistant to hypotonic influences than normalcells.

This new property of altered cells relative to normal cells 2 incubationin a hypotonic solution.

The step of incubation in a hypotonic solution may be followed by apurification step. The non-disintegrated cells are collected in thisstep. The cells thus obtained can then be subjected to analysis. Thisanalysis can, for example, take the form of analysis using RT-PCR.

The analysis of the derived cells can embrace the determination of theexpression of a tumor marker, such as associated/specific mRNA. Inparticular, when this method forms part of a diagnostic method fortumors, such as circulating and micro-metastatic tumor cells.

The method according to the invention enables the separation of cellfractions, comprising cells that are responsible for the backgroundexpression of a tumor marker, from altered cells, such as tumor cells,that may be present in the test sample at a low number. The methodaccording to the invention allows the detection of the presence ofaltered cells, such as tumor cells, in test samples that would notnormally be possible because of the background expression of theselected tumor marker by normal cells. The background expression throughnormal cells can lead to a false-positive signal in the test sample.

The test samples may be mixtures of normal cells and altered cells frombody fluids or tissue. In particular, the body fluid may be one of thefollowing: Blood, urine, cerebrospinal fluid, bone marrow, lymph,ascites or sputum.

As already mentioned above, the altered cells may be tumor cells.Preferred are tumor cells, circulating and/or micro-metastatic tumorcells of solid tumors such as carcinomas in tissues and body fluids,where this type of tumor does not occur.

In particular, the micrometastatic tumor cells are tumor cells ofepithelial origin.

In a preferred embodiment of the invention the test sample is bonemarrow or whole blood.

The normal cells of the test sample, which can express one or moretumor-associated mRNA species are:

-   Cells of the myelotic or lymphatic differentiation series at    different stages of maturity. These include undifferentiated    myeloblasts and stages of maturity through to segmented granulocytes    and monocytes/macrophages; undifferentiated megakaryoblasts and    maturity stages through to thrombocytes; proerythroblasts and    maturity stages through to reticulocytes.

The lymphatic series includes leucocytes of the Iymphatic series atdifferent stages of differentiation, in particular lymphatic stem cellsand maturity stages through to differentiated effector cells of theT-lymphocyte/B-lymphocyte series.

According to the invention these normal cells, or non-tumor cells, thatexpress the associated-associated mRNA either in a quiescent state, oronly in an activated/stimulated state, are eliminated from the sampleonly upon use of a hypotonic solution before the detection of thepresence of tumor cells.

The hypotonic solution has an osmolality below 100 mosm/kg. Thepreferred osmolality of the solution is in the range 30-60 mosm/kg, moreexactly 40 mosm/kg.

The hypotonic solution can be added as such to the cells or with the useof auxiliary agents such as Sephadex, active charcoal or ion exchangersto lower the osmolality of the solution. A preferred hypotonic solutionis based on a solution of salts such as, for example: NaCl, KCl, NH₃Cl,phosphate buffered saline (PBS), Hank's Balanced Salt Solution (HBBS)and mixtures thereof. Alternatively, pure water may be used.

The hypotonic solution can contain further adjuvants that promote thedisintegrating effect of the hypotonic solution or accelerate thedegradation of constituents of the disintegrating cells. These adjuvantscan be ionic and non-ionic tensides such as, for example, saponin,Triton, Tween, sodium dodecyl sulfate (SDS). Further adjuvants areenzymes that degrade nucleic acid (RNases and DNases) and/orprotein-grading enzymes (proteinase K, pronase or others).

In a preferred embodiment, the hypotonic solution contains enzymes thatdegrade nucleic acid and/or protein-degrading enzymes, such as RNase.

In view of the general instability of cellular mRNA, it is advantageousfor the elimination of non-tumor cells to be carried out rapidly anddirectly after sampling and at the same location. The short lapse oftime ensures that the integrity of the test sample is maintained andalso enables the presence of small quantities of tumor cells in thesample to be detected (diagnostic sensitivity).

According to the invention, the elimination (destruction) is carried outthrough incubation of cells of the test sample in a hypotonic solution.The constituents of the cells, such as RNases, released in the process,or artificially-added constituents, such as enzymes, including RNases,can degrade the damaged or lysed cells, and thus, in particular, removetheir mRNA - and thereby the illegitimate mRNA transcription products.

The invention provides for a subsequent so-called stabilization of thesample. This stabilization step can be performed before or afterrecovery of the non-destroyed cells. If stabilization is carried outafter the recovery of the non-destroyed cells then the solution willresult in the lysis of all remaining cells, including the altered cells,in particular tumor cells, with concurrent stabilization of the mRNA ofthose cells. With the lysis it must be ensured that free enzymes thatexhibit a nucleic acid-degrading activity, are deactivated so that theydo not destroy the associated-associated mRNA of the altered cells, suchas tumor cells, released at the same time.

This solution can contain a highly-concentrated chaotropic salt (forexample guanidinium isothiocyanate or guanidinium hydrochloride) forstabilization of the RNA.

The tumor-associated/specific mRNA, the presence of which isdemonstrated in the analysis of the derived cells, may be selected fromcytokeratin 18 (CK18), cytokeratin 19 (CK1 9) and cytokeratin 20 (CK20),as well as other members of the cytokeratin family, carcinoembryonicantigen (CEA), ErbB2, ErbB3, epithelial mucin-1, epithelial mucin-18,guanylyl cyclase C, Cdx-1, Cdx-2, prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), sucrose isomaltase, lactase,carbonic anhydrase, tyrosinase, thyroglobulin, tyrosine hydroxylase,neurone-specific glycoprotein, desmoplakin l, epithelial glycoprotein 40or gastrointestinal tumor-associated antigen.

A potentiation of the lysis effect of the hypotonic solution on cellfractions of blood is possible through the use of specific antibodiesagainst the cell fractions to be destroyed through hypotonic lysis,coupled with the use of complement. Cells, for example, thegranulocytes, are pre-damaged in this process by antibodies directedagainst surface antigens, e.g. CD123, CD125 and complement in a way thata less hypotonic solution (>100 mosm/kg) is sufficient in a followingstep to lyse the target cells. A pre-requirement is that the antibodiesused are capable of triggering complement lysis.

Antibodies that are not able to mediate complement lysis can, however,similarly be used in an embodiment form according to the invention tosupplement the system. Such antibodies are directed against surfaceantigens on the tumor cells, to which they bind, and stabilize them, sothat the tumor cells to be detected also remain intact at a very lowosmolality (<15 mosm/kg).

It is clear that the method according to the invention can be used incombination with known methods.

The kit according to the invention for the detection of tumor cells in asample comprises a hypotonic solution and primer for detecting thepresence of mRNA coding for a marker for altered cells, such as tumorcells. This marker can take the form of a tumor-associated/specificmRNA. If the analysis embraces the detection of mRNA, the kitadvantageously contains a RNA-stabilizing solution, comprising ahighly-concentrated chaotropic salt.

The hypotonic solution, or means to bring about hypotonic conditions, scontained in the kit results in an osmolality below 100 mosm/kg. Thepreferred osmolality range of the hypotonic solution is 30-60 mosm/kg.

The kit according to the invention can, in particular, be used underroutine diagnostic conditions for the diagnosis of metastatic cancer.The presence of tumor cells in the test sample is detected without afalse-positive signal resulting from the illegitimate expression of themarker by normal cells.

The invention is described below in more detail through examples. Theseexamples, however, are in no way limiting for the invention.

EXAMPLE 1. Production and Measurement of Defined Hypotonic Solutions

Defined hypotonic solutions:

Osmolality was determined using the following apparatus: FiskeOsmometer, model 2400 Multisample (Dr. Berthold G. SchlagWissenschaftliche Messinstrumente Nachf. GmbH, Am Muhlenberg 19, D-51465Bergisch Gladbach, Germany). This is a commonly-used instrument fordetermination of osmotic pressure through depression of the freezingpoint. The instructions of the manufacturer were followed.

Serial dilutions of PBS (Phosphate Buffered Saline, BioWhittaker,BE17-516F, 0.0067 M (PO₄)) were prepared. Undiluted solution wasassigned a value of 100%. Various hypotonic solutions were produced andtheir osmolality determined (P-solutions), see Table 1. TABLE 1 % PBSOsmolality (mosm/kg) P100 287 P35 103 P30 84 P25 72 P20 58 P15 42 P10 26P5 13 P1 2

Serial dilutions of HBSS (Hank's Balanced Salt Solution, (PAA, Cat No.H15-012) were prepared. Undiluted solution was assigned a value of 100%.Various hypotonic solutions were produced and their osmolalitydetermined (H-solutions), see Table 2. TABLE 2 % HBSS Osmolality(mosm/kg) H100 276 H50 147 H35 101 H30 86 H20 56 H15 46 H10 24 H5 11 H10

The osmolality of the solutions was determined as follows:

Sample volume: A sample volume of 20 μl, calibrated for measurement, wasintroduced into the measurement cell of the instrument. Measurement wascarried out using appropriate internal quality controls to ensure goodprecision of the measurements.

Procedure:

20 μl of each different dilution of an isotonic starting solution waspipetted in its entirety, without bubble formation, onto the base of thesample vessel in a sample ring. Measurement was carried out fullyautomatically for up to 20 samples and the measured values printed outthrough the integrated printer.

Reference Example

A) Blood cells alone:

Blood from volunteer test subjects was used. Coagulation was preventedby adding lithium-heparin in advance.

1. 2 ml heparin-Li whole blood was distributed over the reaction vessels(with 18 ml of the corresponding H/P solution introduced into eachreaction vessel in advance). The solutions were mixed through rotationand incubated at room temperature for 15 minutes.

2. The cells were centrifuged at 350× g, the supernatant discarded andthe pellet was resuspended in 10 ml lysis buffer (R&D Systems, Cat No.WL1000). Mixing was performed by means of shaking, not vortexing.

The first step ensures the elimination of the cells responsible for the“false-positive” background through hypotonic shock. In the second stepthe remaining cells (altered cells/tumor cells) are recovered.

Commercial standardized systems are available for isolation of RNA fromthis step onwards and are mainly based on the method of Chomczynski andSacchi (Anal Biochem. 1987 Apr; 162 (1): 156-9). In the case describedhere, the RNA was isolated using a kit from the company Qiagen (QlAmpRNA Blood Mini Kit, Cat. No. 523003, Qiagen, Hilden). The instructionsof the manufacturer were followed.

3. The isolated RNA was eluted from the membrane with 30μl ddH₂0 andconcentrated in a vacuum centrifuge. The pellet was then resuspended in13 μl ddH₂O.

4. 3 μl was used for determination of the RNA concentration.

5. 10 μl RNA was used for the cDNA synthesis. cDNA synthesis is aconstituent part of the Roche CK20 PCR kit (Cat. No. 3118 835) and wasperformed in accordance with the instructions of the manufacturer.

6. 4μl of the cDNA obtained was introduced into the CK-20 LightCyclerPCR and the instrument used in accordance with the instructions of themanufacturer.

7. The studies were evaluated using LightCycler Software 3.5.

B) Blood cells with admixed tumor cells:

1. HT29 colon carcinoma cells were separated from their substrate usingAccutase™ (PAA-Laboratories GmbH, Cat No. L11-007) at a volume of 3m1per T175 culture flask. Cell culture medium (RPMI 1640+10% FCS+2mMglutamine+1 mM sodium pyruvate, Invitrogen, Life Technologies Karlsruhe)was then added to make the volume up to 30 ml to inactivate the Accutaseand the cells were centrifuged (1400 rpm for 5 min.). The pellet wasresuspended in 20 ml and the cell count was determined using a Neubauercell counting chamber. The desired cell number per ml was then derivedby further dilution with culture medium.

If >100 cells per run were added per aliquot, then the cell number wasadjusted through dilution. For cell numbers <100, the tumor cells werecounted out of the resuspended tumor cell suspension using amicromanipulator and transferred to a reaction vessel in which 100 μlPBS or HBSS was introduced in advance. The cells were admixed with theblood through the addition of 100 μl prior to the blood (2ml) with thecorresponding P/H solutions.

The further steps in the procedure and the evaluation were as describedunder A) 1-7.

Example 1

Blood was taken from a blood donor and divided into 2 aliquots. Thefirst aliquot was treated on its own and for the second aliquot afteraddition of 1000 HT29 tumor cells. Since only 20% of the cDNA generatedcan be used for the CK-20 PCR, the signals correspond to those for 200HT29 cells. The samples were treated and analyzed in the same manner asthe reference example.

FIG. 1 a shows the results for the test run without addition of tumorcells. CK20 and PBGD signals are detected for the blood cells treatedwith a solution of 276 mosmoles (H100). No CK20 signal is seen aftertreatment with H solutions (H15-HO) with a lower osmolality (<46mosm/kg), but the PBGD signal is present (sufficient sample material wastherefore present).

FIG. 1 b shows the results for the test run with tumor cells added(HT29). CK20 and PBGD signals are also detected after treatment of thesample with solutions of low and very low osmolality (H15, HO). Since noCK20 signal was seen in the parallel test run without addition of tumorcells at an osmolality below 46mosm/kg, these signals are clearlyattributable to the added tumor cells.

The results shown in the Figures are representative extracts fromseveral test runs with different hypotonic solutions. The results ofexperiments that go beyond the test runs shown in the Figures aresummarized in Tables 3 and 4. TABLE 3 Positive detection of the Positivedetection of the CK20 signal PBGD signal (Blood cells alone) (Bloodcells alone No. of positive detections/ No. of positive detections/ Hsolution No. of test runs No. of test runs H100 6/6 6/6 H20 2/6 6/6 H150/8 8/8 H10 0/4 4/4 H5 0/3 3/3 H1 0/3 3/3 H0 0/4 4/4

-   -   Table 3: Detection of the CK20/PBGD signal in blood cells as a        function of the hypotonic salt solutions used (H solutions).

The use of a 56-mosmolar solution (H20) led to a 60% elimination of theCK20 signal, with 100% detection of the PBGD signal. A 100% eliminationof the CK20 background signal was achieved with a solution ofapproximately 42 mosmoles (H1 5). The PBGD signal was also 100%retained. TABLE 4 Positive detection of Positive detection of the CK20signal the PGBD signal (Blood cells + HT29 (Blood cells + HT29 tumorcells) tumor cells) No. of positive detections/ No. of positivedetections/ H solution No. of test runs No. of test runs H100 3/3 3/3H20 3/3 3/3 H15 3/3 3/3 H0 3/3 3/3

-   -   Table 4: Detection of the CK20/PBGD signal in blood cells with        added tumor cells as a function of the hypotonic salt solutions        used (H solutions).

Both the CK20 and the PGBD signal were seen under all test conditions.This means that tumor cells are resistant to the hypotonic solutionsused and thus can be differentiated from the blood cells.

Further studies, in which a low number of tumor cells were added, showedthat CK20 signals could be detected for 5 tumor cells.

Example 2

Blood was taken from a blood donor and split into 2 aliquots. The firstaliquot was used without addition of tumor cells the second aliquot wasmixed with 25 HT29 tumor cells.

The samples were prepared and analyzed in the same manner as thereference example.

Since only 20% of the cDNA generated can be used for the CK-20 PCR, thes signals correspond to those for 5 HT29 cells. For this donor, thebackground signal was reached with hypotonic solutions at an osmolalityof just 103 mosm/kg.

FIG. 2 shows the results of quantitative RT-PCR for CK20 and PBGD. TheCK20 and PBGD signals can be detected without treatment of the bloodcells with hypotonic solutions (blood alone). Incubation with a P35solution (103 mosm/kg) leads to elimination of the CK20 signal. The PBGDsignal is retained (with P35). In the parallel run, containing 25 HT29tumor cells, the CK20 signal remains detectable (blood+25Ht29+P35).

Example 3 Improvement of the Results of Lysis Through Addition of RNaseA

Example 1 was repeated. RNase A (1 mg/aliquot) was added to the bloodbefore treatment with the P/H solution to enable rapid degradation ofthe RNA before the final lysis through the RLT buffer containingguanidinium isothiocyanate. This led to an improvement in the results oflysis, i.e., lysis was also possible with solutions of higherosmolality.

Example 4 Primary Effect of P/H Solutions on Granulocytes as a CellFraction of Normal Cells in the Test Sample

The finding that the CK20 signal is expressed by the granulocytefraction of the leukocytes, underlines the results presented here. FACSanalysis was carried out to investigate the composition of the bloodcell fractions, with and without P/H solutions, and a marked reductionin the number of granulocytes was observed in agreement with the CK20data.

The most marked reduction was observed at an osmolality of 25-40mosm/kg.

The test procedure was as follows:

-   -   1. 4 ml heparin blood was mixed with 100ul antibody solution        directed against the surface antigen CD45/CD14 (Simultest        LeucoGate, BD-Biosciences, Cat. No. 342408) and CD16 (Caltag,        Cat. No. MHCD1606) in accordance with the instructions of the        manufacturer and incubated at room temperature (RT) for 15        minutes.    -   2. 24 FACS tubes, each containing 2 ml H-solution, were        prepared.    -   3. 150μl blood was added to each 2 ml of H-solution and mixed        well, followed by incubation for 15 minutes at room temperature.    -   4. The tubes were centrifuged for 5 minutes at 300× g.    -   5. The pellet was resuspended in 2-3 ml erythrocyte lysis buffer        (R&D Systems Inc., Cat. No. WL1000).    -   6. The samples were incubated for 10 minutes at room        temperature.    -   7. The samples were centrifuged for 5 minutes at 300× g.    -   8. The pellet was washed in 2-3 ml wash buffer (R&D Systems        Inc., Cat. No. WL1000).    -   9. The sample was again centrifuged for 5 minutes at 300× g and        washed with 5ml Cellwash (BD Biosciences Cat. No. 349524). It        was then re-centrifuged for 5 minutes at 300× g.    -   10. The pellet was resuspended in 500 μl Cellwash (BD        Biosciences Cat. No. 30 349524), and 60 μl fixative (R&D Systems        Inc., Cat. No. WL1000) added    -   11. FACS measurement with immunologically-differentiated surface        was performed with a FACSCalibur flow cytometer (BD Biosciences,        Germany).

FIG. 3 shows the relative proportion of granulocyte subpopulations ofblood cells within the blood cells as a function of treatment withhypotonic solutions. A reduction in the granulocyte population can beseen that is dependent on the treatment with hypotonic solutions. Thisreduction is very drastic for solutions in the range 20-40 mosm/kg. Thepercentage of granulocytes relative to the total cell number fell inthis range from approximately 66% to approximately 13%. For solutions<20 mosm/kg, the percentage of granulocytes stabilized at a level ofapproximately 10% of the total cell number.

Three independent experiments are shown, indicated by different symbols.

1. A method for the separation of cell fractions, comprising normalcells and altered cells, comprising the step of incubating the mixtureof normal cells and altered cells in a hypotonic solution and thedestruction of one or more cell fractions thereof.
 2. A method accordingto claim 1, further comprising the subsequent step of collecting thenon-destroyed cell fraction.
 3. A method according to claim 1, furthercomprising the subsequent step of analysis of the cells of the collectedcell fractions.
 4. A method according to claim 3, wherein the analysisof the derived cells is through polymerase chain reaction (PCR).
 5. Amethod according to claim 1, wherein the mixture of normal cells andaltered cells is derived from bodily fluids or tissue.
 6. A methodaccording to claim 5, where the bodily fluid is selected from the groupcomprising blood, urine cerebrospinal fluid, bone marrow, lymph, ascitesand sputum.
 7. A method according to claim 1, wherein the altered cellsare tumor cells.
 8. A method according to claim 1, wherein the tumorcells are circulating and/or micrometastatic tumor cells.
 9. A methodaccording to claim 1, wherein the normal cells are only mononuclearcells from the blooc.
 10. A method according to claim 1, wherein thetumor cells are selected from the group consisting of the group of solidmalignant tumors of epithelial origin (carcinomas).
 11. A methodaccording to claim 1, wherein the osmolality of the hypotonic solutionis below 100 mosm/kg.
 12. A method according to claim 1, wherein thehypotonic solution is a salt solution selected from the salts NaCl, Kcl,NH₂Cl, Phosphate Buffered Saline (PBS), Hank's Balanced Salt Solution(HBBS) or mixtures thereof.
 13. A method according to claim 1, whereinthe hypotonic solution firther contains enzymes that degrade nucleicacid and/or protein-degrading enzymes.
 14. A method according to claim1, wherein the hypotonic solution further contains Rnase.
 15. A methodaccording to claim 3, wherein the analysis of the derived cellscomprises the determination of the expression of a tumor marker.
 16. Amethod according to claim 15, wherein the tumor marker is selected fromcytokeratin 18 (CK18), cytokeratin 19 (CK19), cytokeratin 20 (CK20) andfurther members of the cytokeratin family, carcinoembryonic antigen(CEA), ErbB2, ErbB3, epithellal mucin-1, epithelial mucin-18, guanylylcyclase C, Cdx-1, Cdx-2, prostate specific antigen (PSA), prostatespecific membrane antigen (PSMA), sucrose isomaltase, lactase, carbonicanhydrase, typrosinase, thyroglubulin, tyrosine hydroxylase, neuronespecific glycoprotein, Desmoplakin 1, epithelial glycoprotein 40 andgastrointestinal associated-associate antigen.
 17. Use of a methodaccording to claim 1, to detect the presence of altered cells, inparticular tumor cells.
 18. Use of a method according to claim 1, forthe diagnosis of metastatic cancer.
 19. Kit to detect the presence oftumor cells in a sample, comprising a) a hypotonic solution, and b)primer to detect the presence of MRNA coding for a tumor marker.
 20. Kitaccording to claim 19, further comprising c) an RNA-stabilizingsolution, comprising a highly-concentrated chaotropic salt.
 21. Kitaccording to claim 19, wherein the hypotonic solution has an osmolalitybelow 100 mosm/kg.
 22. Kit according to claim 19, wherein the hypotonicsolution is a salt solution, selected from the salts NaCl, Kcl, NH₃Cl,Phosphate Buffered Saline (PBS), Hank's Balanced Salt Solution (HBBS)and mixtures thereof.
 23. Kit according to claim 19, for the diagnosisof matastatic cancer.
 24. Kit according to claim 19, wherein the markeris selected from the group comprising cytokeratin 18 (CK18), cytokeratin19 (CK19), cytokeratin 20 (CK20) and firther members of the cytokeratinfamily, carcinoembryonic antigen (CEA), ErbB2, ErbB3, epithelialmucin-1, epithelial mucin- 18, guanylyl cyolase C, Cdx- 1, Cdx-2,prostate specific antigen (PSA), prostate specific membrane antigen(PSMA), sucrose isomaltase, lactase, carbonic anhydrase, tyrosinase,throglubulin, tyrosine hydroxylase, neuron-specific glycoprotein,Desmoplakin 1, epithelial glycoprotein 40 and gastrointestinalassociated, associated antigen.
 25. Use of a kit according to claim 19,to detect the presence of tumor cells in a sample.